Matrix metalloproteinases (MMPs; matrixins) comprise a family of structurally related Zn-containing proteases that degrade all macromolecules present in the extracellular matrix (ECM). Each of the known MMPs can be divided up into a variable number of well-conserved domains. All contain a pro-peptide which is involved in suppressing the activity of the pro-enzyme form of the molecule, and a HEXXH sequence motif that has been shown by X-ray crystallography to form part of the metal-binding site (Nagase et al. (1999) J. Biol. Chem., 274, 31, 21491–21494). In addition, fibronectin-, hemopexin-, or vitronectin-like domains and/or a membrane “anchor” domain may also be present.
Today, the MMP family includes more than 15 members (Table 1).
TABLE 1Characteristics of known human MMPsMMPTypeSubstrateMMP-1Collagenasecollagen I, II, III, VII, X gelatinsMMP-2Gelatinase Bcollagens IV, V, VII, XI, fibronectin,elastinMMP-3Stromelysin 1proteoglycans, gelatins, fibronectin,collagens II, IV, IXMMP-7MatrilysinProteoglycans, gelatins, collagen IV,elastinMMP-8Neutrophil collagenasecollagens I, II, IIIMMP-9Gelatinase Bgelatins, collagen IV, V,proteoglycan, elastinMMP-10Stromelysin 2proteoglycan, fibronectin, lamininMMP-12Macrophage elastaseproteoglycans, elastaseMMP-13Collagenase 3collagens I, II, III, IV
MMPs are believed to play a critical role in many physiological and pathological processes. The breakdown of ECM by MMPs is essential for processes including embryonic development, morphogenesis, reproduction, and tissue repair and remodeling. Other physiological processes which involve MMPs include tumor growth, tumor invasion, Sjögren's syndrome, periodontal diseases, arthritis, cardiomyopathy, renal failure, atherosclerosis, insulin resistance, adipogenesis, retinal neovascularization, wound healing, and neurodegenerative diseases including, for example, Alzheimer's disease, multiple sclerosis, Parkinson's disease, and motoneuron disease. Identification of the functions of additional genes of the MMP family would be invaluable to those of skill in the art seeking to understand the genetic basis for these processes, as well as identifying compounds that modulate the activity of such genes useful in methods for treating the pathologies.
Tissue inhibitors of metalloproteinases (TIMPs) are a group of closely related secreted proteins that limit MMP activity. To date, four TIMPs have been characterized; TIMP1, TIMP2, TIMP3, and TIMP4, respectively (Gomez et al. (1997) Eur. J. Cell. Biol., 74, 111–122). Several investigators have studied effects of MMP inhibition by using cells over-expressing TIMPs. The balance between MMPs and TIMPs seems to play an important role in matrix turnover in several organ systems.
The past few years have witnessed several advances in the understanding of the pathophysiology of coronary atherosclerosis. The earliest atherosclerotic lesion, named the fatty streak, represents a dynamic balance of the entry and exit of lipoprotein as well as the development of extracellular matrix. A decrease in lipoprotein entry will probably result in a predominance of lipoprotein exit and final scarring. However, an increase of lipoprotein entry can predominate over the efflux and scarring, resulting in vulnerable lipid-rich plaques that are prone to disruption (Falk et al., (1995), Circulation, 92:657–671; Fuster et al., (1999), Lancet, 353:SII: 5–9).
It is evident from many studies that MMPs, as a family, are important regulators of atherosclerotic plaque growth (Newby et al., (1994), Basic Res. Cardiol. 89 [Suppl. 1] 59–70). However, the roles of the individual MMPs are so far largely unknown. Several MMPs are expressed in the diseased blood vessel, i.e. in smooth muscle cells and in macrophages. MMPs likely regulate both the degradation of extracellular matrix and influence the proliferation rate of smooth muscle cells. Several inflammatory cytokines and growth factors increase the expression of MMPs in cell cultures, e.g. interleukin-1, platelet-derived growth factor (PDGF) and tumor necrosis factor-α (TNF-α).
It has been demonstrated in several animal models that inhibition of MMPs (type 1 and 2 among others) decreases smooth muscle proliferation in response to vascular damage. Moreover, MMPs seem to enhance smooth muscle cell migration. These two physiological processes are hallmarks of the neointimal thickening that characterizes atherosclerosis. Accordingly, MMP inhibitors may delay or prevent spontaneous atherogenesis as well as restenosis. MMPs and/or TIMPs may be especially useful for patients at risk for atherosclerosis, dyslipidemia, end-stage renal failure, or patients who have undergone Percutaneous Transluminal Coronary Angioplasty Procedure (PTCA).
A large number of studies support a role of MMPs in intima media function. For example, over-expression of TIMP2 inhibits vascular smooth muscle cell proliferation and chemotaxis in vitro (Baker et al., (1998), J. Clin. Invest., 101: 1478–1487; Cheng et al., (1998), Circulation, 98:2195–2201). In addition, it has been shown that MMPs are linked to the proliferation and outgrowth of vascular smooth muscle cells from explants of rabbit aorta in vitro. The proliferation and outgrowth of vascular smooth muscle cells from rabbit aorta was blocked by experimental inhibitors (Ro 31-4724 and Ro 31-7467) (Newby et al., (1994). Batimastat (BB94), a synthetic MMP inhibitor, can reduce smooth muscle cell proliferation in vitro as well as inhibit neointimal formation after balloon injury to the rat carotid artery (Zempo et al., (1996), Artherioscler. Thromb. Vasc. Biol., 16:28–33). Local overexpression of TIMP1 has been shown to inhibit intimal hyperplasia in rats (Forough et al., (1996), Circ. Res., 79:812–820). After in vitro incubation with MMP-3, -7, or -12, the ability of HDL(3) to induce the high affinity component of cholesterol efflux from the macrophage foam cells was strongly reduced (Lindstedt et al., (1999), J. Biol. Chem., 274:22627–22634).
Angiogenesis, also known as neovascularization or new vessel growth, is part of the normal wound healing machinery and can occur as a reaction to tissue hypoxia. Various tumors are also known to trigger angiogenesis, leading to tumor growth. In normal adult tissue, there is a balance between angiogenic and anti-angiogenic factors and, as a result, few new vessels are formed. However, if the balance between angiogenic and anti-angiogenic factors is disturbed, a complex cascade of events can be triggered that eventually leads to the formation of new blood vessels.
Diabetic retinopathy is the leading cause of blindness for the majority of Americans. Microvascular damage from diabetes leads to microaneurysms, hemorrhage, exudates, and cotton-wool spots. Further progression of disease leads to neovascularization. Growth of new blood vessels can cause severe hemorrhage, scarring, and permanent visual loss (for a review, see Frank et al, (1996), South. Med. J., 89:463–470; Jampol & Goldbaum, (1980), Surv. Ophthalmol. 25:1–14). Various randomized, prospective studies have clearly shown benefit from laser therapy at specific stages of progression of retinopathy.
AMD with rapid progression (wet AMD) is another common cause of blindness in the developed world. Presently the underlying etiology of AMD is unknown but a slow deterioration of the retinal pigment epithelium, leading to the death of macular photoreceptors, is believed to be an important factor. The wet form of AMD often leads to a complete loss of central vision within a few years. AMD usually debuts in the dry form and may subsequently change into the wet form. AMD with rapid progression is characterized by choroidal new vessel formation (CNV). The new vessels tend to leak and may rupture. The resulting macular edema, bleeding, fibrinous deposits, and scar formation are reasons for the rapid deterioration of vision in this form of AMD.
Sprouting is a key step in CNV formation. If sprouting can be inhibited, no new leaky vessels will form. MMPs are essential to create space for the new sprouts. Because this step is downstream in the angiogenesis process, an MMP inhibitor can work to limit sprouting even if the earlier events are slightly different from those described above. The localization of MMP-2 and MMP-9 to the areas of new vessel formation and to the enveloping Bruch's-like membrane, respectively, suggests that MMP-2 and MMP-9 may be cooperatively involved in the progressive growth of choroidal neovascular membranes (Steen et al. (1998), Invest. Ophthalmol. Vis. Sci., 39:2194–2200). In normal individuals MMP-9 activity is not detected in the eye; however, it has been demonstrated that MMP-9 activity is detected in more than 80% of patients with “active” proliferative retinopathy (Kosano et al., (1999), Life Sci., 64:2307–2315).
MMP inhibitors present an attractive opening for prophylactic pharmacotherapy of ocular blood vessel proliferation in diabetes and AMD. Moreover, it may be possible to combine MMP inhibitors with photodynamic therapy. It is possible that inhibitors of MMPs could prevent recurrence of CNV and, thus, improve long-term efficacy. Patients are likely to accept certain side effects in order to preserve their vision, as most are aware that the disease will rapidly lead to blindness. Topical treatment is advantageous from a pharmacovigilance point-of-view.
MMPs are also involved in the bioacticvation of cytokines, including tumor necrosis factor-alpha (TNF-α). Evidence suggests that TNF-α is a key mediator of insulin resistance in adipocytes and skeletal muscle. Inhibition of MMPs may decrease the formation rate of TNF-α and, accordingly, be of therapeutic significance in type-II diabetes. MMPs and/or TIMPS may be useful for patients with Type II diabetes or for obese patients with insulin resistance.
It has been suggested that TNF-α is an inducer of insulin resistance in type II diabetes. TNF-α is synthesized as a membrane-bound precursor that is proteolytically processed to an active form by a matrix metalloproteinase (MMP)-like enzyme. It has been shown that subcutaneous administration of KB-R7785 (a non-specific MMP inhibitor) to KKAy mice, which show insulin resistance and hyperglycemia for 4 weeks, resulted in a significant decrease in plasma glucose levels after 3 weeks of administration. In the same study it was also demonstrated that administration of pioglitazone significantly decreased plasma glucose levels. Interestingly, KB-R7785, but not pioglitazone, also significantly decreased plasma insulin levels in the animals. It has also been shown that the lipopolysaccharide-induction of TNF-α in plasma can be inhibited in KKAy mice by KB-R7785. These results suggest that MMP inhibitors may exert an anti-diabetic effect by ameliorating insulin sensitivity through the inhibition of TNF-α production.
Nephropathy in patients with type I and II diabetes mellitus is a rapidly increasing problem worldwide. Diabetic patients account for nearly half of all patients on hemodialysis. Microalbuminuria is diagnosed when the urinary albumin excretion rate is greater than 20 but less than 200 micrograms/min and the prevalence of microalbuminuria among diabetic patients is 15–20% (Deckert et al., (1992), Diabetes Care, 15:1181–1191).
MMP inhibitors (non-selective) have been found to decrease the proliferation rate of cultured rat mesangial cells without affecting cell viability. Therefore, MMP inhibitors may offer a new therapeutic approach for treatment of mesangial cell-derived forms of glomerulonephritis and prevent basal membrane thickening in diabetes. MMPs and/or TIMPS may be useful for diabetic patients with early signs of glomerulopathy, or for patients with microalbuminuria.
Progressive expansion of the mesangial matrix, and thickening of the glomerular and tubular basement membranes are hallmarks of human and experimental diabetic nephropathy (Philips et al. (1999), Kidney Blood Press. Res. 22:81–97; Young et al., (1995) Kidney Int., 47:935–944). These lesions eventually lead to glomerular fibrosis, a central pathological feature in many human acute and chronic kidney diseases, which progressively destroys the renal filtration unit, and may finally cause renal failure. It has been demonstrated that mesangial matrix expansion is strongly related to the clinical manifestation of diabetic nephropathy. Diabetic nephropathy is effected both directly and indirectly by the alteration of cytokine generation. Data from studies on several animal species suggest that proliferation of mesangial cells is an important feature of diabetic glomerulopathy. Harendza et al., (Nephrol. Dial. Transplant 12:2537–2541, (1997)) have demonstrated that the expression of MMP2 is enhanced in experimental proliferative glomerulopathy in the rat. Inhibition of MMP2 by Ro 31-9790 inhibited the proliferation rate of cultured rat mesangial cells in a concentration-dependent and at least partially reversible manner without affecting cell viability (Steinmann-Niggli K, et al., (1997), J. Am. Soc. Nephrol. 8:395–405). Moreover, Ebihara et al., ((1998) Am. J. Kidney Dis. 32:544–550) have reported that increased MMP9 concentrations in plasma preceded the occurrence of microalbuminuria in diabetic patients.
Thus, a need exists for new members of the MMP family of proteases.